Maintaining electrolyte homeostasis is critical for preserving a cell's ability to interact with other cells and transduce signals that are mediated by changes in the environment. The levels of Ca.sup.++ and P.sub.i in extracellular fluid is tightly regulated, and excitability of nerve and muscle are dependent upon ion concentration. In vertebrates parathyroid hormone (PTH), active metabolites of vitamin D and calcitonin have been identified as regulators of plasma levels of Ca.sup.++ and P.sub.i. PTH stimulates the mobilization of Ca.sup.++ and P.sub.i from bone into plasma. In the kidneys, PTH decreases the urinary excretion of Ca.sup.++ and stimulates excretion of P.sub.i. P.sub.i reabsorption is reduced by decreasing the Na.sup.++ -dependent P.sub.i transport into the renal proximal tubule. Calcitonin is a hormone released by the thyroid that lowers Ca.sup.++ and P.sub.i levels in the blood to maintain the equilibrium when serum levels are elevated. Calcitonin is believed to counteract hypercalcemia by inhibiting osteoclast-mediated bone resorption. However, the role of calcitonin as regulator of transient influcuations in serum calcium levels or promotion of bone mineralization has not been demonstrated. In addition, there are no known regulators of transient changes P.sub.i levels in mammals, other than PTH, which only promotes P.sub.i excretion, thereby suggesting that our understanding and identification of factors involved in the calcium and phosphate homeostasis is incomplete.
In fish, the environment can be either hypertonic (seawater) or hypotonic (fresh water) and is the primary source for Ca.sup.++ and P.sub.i, unlike terrestrial vertebrates whose uptake of Ca.sup.++ and P.sub.i are diet dependent. Some fish migrate to and from seawater and fresh water and have had to develop mechanisms to adapt to these radical changes in electrolyte concentrations. Stanniocalcin (also known as Stc, CS protein, teleocalcin or hypocalcin) is a homodimeric glycoprotein hormone secreted by endocrine glands found on the kidneys of bony fish called the corpuscles of Stannius (Stannius, H. Arch. Anat. Physiol. 6:97-101, 1839 and Wagner, G., in Biochemistry and Molecular Biology of Fishes, eds. Hochachka and Mommsen, Elsevier Science, Amersterdam, Vol 2, Chap. 21, pp. 419-434, 1993). The secretion of stanniocalcin in fishes is stimulated in response to rising plasma Ca.sup.++ levels (Wagner et al., Mol. Cell Endocrinol. 79:129-138, 1991) whereupon it acts to reduce Ca.sup.++ uptake by the gills (Wagner et al., Gen. Comp. Endocrinol. 63:481-491, 1986) and increase P.sub.i reabsorption by the kidneys (Lu et al., Am. J. Physiol. 36:R1356-R1362, 1994) with the net result being restoration of normal calcium levels.
Stanniocalcin has been identified and isolated from several species of fish, such as Australian eel (Auguilla australis; Butkus et al., Mol. Cell. Endocrinol 54:123-134, 1987), rainbow trout (Oncorhynchus mykiss; Lafeber et al., Gen. Comp. Endocrinol. 69:19-30, 1988), coho salmon (Oncorhynchus kisutch; Wagner et al., Mol. Cell. Endocrinol. 90:7-15, 1992), and sockeye salmon (Oncorhychus nerka; Wagner et al., Gen. Comp. Endocrinol. 72:237-246, 1988. Studies using both in vivo and in vitro systems, show that in response to increased excellular Ca.sup.++ levels, Stc is released. Stc reduces plasma Ca.sup.++ concentration by inhibiting Ca.sup.++ absorption across the intestine (Sundell et al., J. Compl Physiol. B, Biochem Sys. Environ. Physiol. 162:489-495, 1992) and Ca.sup.++ transport across the gills (Wagner et al., ibid., 1988). In vivo, the rapid equilibration that occurs even when presented with massive increases in Ca.sup.++ suggests that there may be additional pathways involved that have not yet been elucidated.
Human stanniocalcin has recently been discovered (Chang et al., Mol. Cell. Endocrinol. 112:241-247, 1995 and Olsen et al. WO 95/24411). The human protein is 247 amino acids, of which 214 amino acids constitute the mature polypeptide. It is believed that the first 33 amino acids represent a prepro region, consistent with Stc from fish, where the polypeptide is synthesized as a larger molecule and processed during secretion from the Corpuscles of Stannius. The human stanniocalcin discovered had a high degree of homology to the Australian eel; 119 identical amino acids in a 195 amino acid overlap (61% identity) and to coho salmon, 118 amino acids in a 204 amino acid overlap resulted in 57% identity. Eel and coho salmon Stc contain 15 and 12 cysteines respectively, while the human Stc has 11 cysteines. The spacing of the cysteines is highly conserved between all three proteins.
Human stanniocalcin mRNA was found to be most abundant in ovary, prostate and thyroid as a 4 kb transcript (Chang et al., Mol. Cell. Endocrinol. 112:241-247, 1995). The 4 kb band was also seen in kidney, bone marrow, thymic stromal cells (Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796, 1996) and many other tissues, but no signal was found in brain, liver, spleen, peripheral blood leukocytes and adrenal medulla. In addition, a 2 kb transcript was identified with a probe designed to a 1 kb N-terminal portion of the molecule but was not seen with a probe containing C-terminal sequence (Chang et al., ibid., 1995). Isolation of human stanniocalcin has been reported from an early stage lung cDNA library (Olsen et al., WO 95/24441). Sera from normal humans was immunoreactive with salmon Stc antibodies. Analyses by immunocytochemistry suggested that cells in the human kidney tubules contained Stc-like proteins (Wagner et al., Proc. Natl. Acad. Sci. USA 92:1871-1875, 1995).
Human stanniocalcin mRNA levels have been shown to increase in the presence of increasing amounts of excellular Ca.sup.++ in an immortalized liver fibroblast cell line SUSM-1, T98G human glioblastoma cell line and normal human foreskin fibroblasts (Chang et al., ibid., 1995).
Because of the anti-hypercalcemic activity of stanniocalcin, members of the family, particularly human polypeptides will be very valuable in the understanding and treatment of diseases caused by electrolyte disorders. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.